1. Volume 11, Issue 3, Pages 828-841 (September 2018)
Cell-Surface Marker Signature for Enrichment of Ventricular Cardiomyocytes Derived from Human Embryonic Stem Cells 
Jennifer Veevers, Elie N. Farah, Mirko Corselli, Alec D. Witty, Karina Palomares, Jason G. Vidal, Nil Emre, Christian T. Carson, Kunfu Ouyang, Canzhao Liu, Patrick van Vliet, Maggie Zhu, Jeffrey M. Hegarty, Dekker C. Deacon, Jonathan D. Grinstein, Ralf J. Dirschinger, Kelly A. Frazer, Eric D. Adler, Kirk U. Knowlton, Neil C. Chi, Jody C. Martin, Ju Chen, Sylvia M. Evans 
Stem Cell Reports 
Volume 11, Issue 3, Pages (September 2018)
DOI: /j.stemcr
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2. Figure 1
Generation of an H9 MYL2-GFP BAC Transgenic Reporter Cell Line
(A) A schematic representation of the BAC targeting vector containing: a histone2B-GFP-IRES-neomycin resistance gene cassette (H2B-GFP-IRES-NeoR) integrated in-frame to the ATG start site of the cardiac ventricle-specific human MYL2 gene, and a PGK-neomycin resistance (PGK-NeoR) gene cassette encoding G418 resistance flanked by loxP sites (black triangles). The predicted sizes of the PCR products using primers (P) 1–4 to detect genomic integration are indicated.
(B) RT-PCR amplification of a 1-kb band (P1 and P3) and a 0.6-kb band (P2 and P4), and long-range PCR amplification of a specific 4.1-kb band (P1 and P2) confirmed BAC construct integration in G418-resistent clones nos 1–6. BAC DNA was used as a positive control. Genomic DNA from non-transfected wild-type H9 hESCs was used as a negative control. M, marker. See Figure S1 for verification of H9 MYL2-GFP BAC reporter line pluripotency and cardiac differentiation capacity.
(C) Flow cytometric quantification of MYL2-H2B-GFP expression over the time course of embryoid body cardiac differentiation (day 0–25). Numbers indicate the percentage of GFP+ cells (see Figure S2A for cell gating strategy). Data are representative of a minimum of three independent clones.
(D) Immunofluorescence analysis (MYL2-H2B-GFP) of a day 25 embryoid body derived from H9 MYL2-GFP hESCs. Scale bar, 100 μm. The image is representative of a minimum of three independent clones.
(E) Immunofluorescence analysis of hESC-derived cardiomyocytes after 25 days of cardiac differentiation. MYL2-H2B-GFP-positive(+) cells (i) were positive for cardiac troponin T (cTNT) (ii). (iii) DNA stained with DAPI. (iv) Merged image. Arrows point to MYL2-H2B-GFP+/cTNT+ cardiomyocytes. Asterisks indicate cTNT+ cardiomyocytes not expressing MYL2-H2B-GFP. Scale bar, 20 μm. Images are representative of a minimum of three independent clones.
(F) Immunostaining for MYL2 (i) shows exclusive expression in MYL2-H2B-GFP+ hESC-derived cardiomyocytes after 25 days of cardiac differentiation (ii and iii). MYL2-H2B-GFP+/MYL2+ cardiomyocytes were positive for α-actinin (vi), but not all α-actinin+ cells (iv) were MYL2+ cardiomyoctes. (v) DNA stained with DAPI. Scale bar, 20 μm. Images are representative of a minimum of three independent clones.
Stem Cell Reports  , DOI: ( /j.stemcr )
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3. Figure 2
Characterization of H9 MYL2-GFP hESC-Derived Ventricular Cardiomyocytes
(A) qRT-PCR analysis of TNNT2 and MYL2 gene expression in unsorted, MYL2-GFP-negative (GFP−)-, SIRPA-bright/MYL2-GFP-negative (SIRPA+/GFP−)-, and MYL2-GFP-positive (GFP+)-sorted fractions of dissociated hESC-derived embryoid bodies at day 25 of cardiac differentiation (see Figure S2 for cell gating strategy). Data were normalized to corresponding ACTB expression, and are relative to the unsorted hESC-derived cell population. Error bars, mean ± SD. ∗∗∗p ≤ by Student's t test (n = 3 technical replicates). Data are representative of a minimum of three independent biological replicates.
(B) Electrophysiological characterization of SIRPA+/GFP+- and SIRPA+/GFP−-sorted fractions of dissociated hESC-derived embryoid bodies at days 25–40 of cardiac differentiation (see Figure S2B for cell gating strategy). (i) Representative triggered action potential (AP) traces recorded from SIRPA+/GFP+ and SIRPA+/GFP− cardiomyocytes using whole-cell current-clamp technique. (ii) Quantification of AP duration (APD) at different times of repolarization (50% and 90%) for SIRPA+/GFP+ (n = 21) and SIRPA+/GFP− (n = 18) cardiomyocytes. Error bars, mean ± SEM. ∗∗p ≤ 0.01 by Student's t test. mV, millivolts; s, seconds. Data are representative of a minimum of three independent biological replicates.
(C) Representative whole-cell currents recorded from (i) SIRPA+/GFP+ (upper panel) and (iv) SIRPA+/GFP− (lower panel) cells, with an enlarged view of the solid boxed area (ii and v), and the dashed boxed area (iii and vi) to the right of the corresponding trace. (vii) Quantification of maximum inward Na+ currents, and maximum sustained K+ currents of SIRPA+/GFP+ (n = 21) and SIRPA+/GFP− (n = 18) cardiomyocytes. Error bars, mean ± SEM. ∗p ≤ 0.05 by Student's t test. Data are representative of a minimum of three independent biological replicates. nA, nanoampere; s, seconds; ms, milliseconds; pA/pF, picoampere per picofarad.
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4. Figure 3
Identification of Positive and Negative Cell-Surface Markers Specific to hESC-Derived Ventricular Cardiomyocytes
(A) After exclusion of debris and doublets based on light scatter properties, single cells were analyzed for the expression of MYL2-GFP. The gating strategy for the detection of MYL2-GFP+ cells was defined using parental H9 cells as a control (see Figure S2A). GFP+ and GFP− populations, represented in green and orange, respectively, were interrogated for the expression of each one of the 242 surface markers included in the BD Lyoplate screening panel.
(B and C) Representative example of positive and negative hits, (B) CD77 and (C) CD200, identified based on median fluorescence intensity (MFI) ratio and differential frequency (%) within GFP+ and GFP− subpopulations. See Figure S3 for other potential marker candidates.
(D and E) Representative heatmaps depicting the highest-ranked positive or negative hits based on (D) MFI ratio or (E) differential frequency (%) within GFP+ and GFP− populations (see Table S1 for heatmap of complete list of 242 markers).
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5. Figure 4
Validation of hESC-Derived Ventricular Cardiomyocyte Cell-Surface Marker Signature
Multi-color flow cytometry was used to simultaneously analyze the expression of surface markers CD77 and CD200, the intracellular cardiomyocyte marker, cardiac troponin I (cTNI), and GFP at day 25 of cardiac differentiation. Data are representative of a minimum of three independent biological replicates.
(A) MYL2-GFP-positive (green) and -negative (orange) populations were defined within single cells, after exclusion of doublets and debris (left panel). The simultaneous expression of CD200 and CD77 was analyzed within the GFP− population (middle panel, orange) and the GFP+ population (right panel, green). See Figure S4 for co-expression of CD77 or CD200 with other marker candidates.
(B) CD77 and CD200 expression was first analyzed within the single-cell population (left panel). The expression of cTNI and MYL2-GFP was then analyzed within the CD77−/CD200+ population (middle panel, orange) and CD77+/CD200− population (right panel, green).
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6. Figure 5
CD77 Expression Highly Correlates with hESC-Derived Ventricular Cardiomyocyte Phenotype
All analysis is of the same population. Data are representative of a minimum of three independent biological replicates.
(A) Correlation of cTNI and (i) CD77, (ii) SIRPA, or (iii) MYL2-GFP in hESC-derived populations at day 25 of cardiac differentiation by flow cytometry.
(B–E) Representative plots depicting cTNI/MYL2-GFP cell populations (right panel, green plots) at day 25 of cardiac differentiation, after gating on different cell populations defined by distinct surface marker signatures: (B) SIRPA+/CD90−; (C) SIRPA+/CD140b−; (D) SIRPA+/CD200−; and (E) CD77+/CD200− (left panel, boxed area).
(F) Left panel: SIRPA+/CD77− (yellow) and SIRPA+/CD77+ (green) cell populations were defined within single cells. Right panel: histograms depicting MYL2-GFP expression within SIRPA+/CD77− and SIRPA+/CD77+ cell populations.
Stem Cell Reports  , DOI: ( /j.stemcr )
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7. Figure 6
Validation of Ventricular Cardiomyocyte Enrichment from hESC-Derived Populations with a Cell-Surface Signature
(A) qRT-PCR analysis of TNNT2, MYL2, MYH6, MYH7, GJA5, and NPPA gene expression in SIRPA+/CD77−-, CD77−/CD200+-, and CD77+/CD200−-sorted fractions of dissociated hESC-derived embryoid bodies at day 25 of cardiac differentiation. Data were normalized to corresponding ACTB expression, and are relative to H9 hESC-derived embryoid bodies at day 0. Data are representative of a minimum of three independent biological replicates.
(B) Immunofluorescence analysis of H9 hESC-derived CD77+/CD200−-sorted cells after 25 days of cardiac differentiation. Cardiomyocytes were stained with cTNT. DNA was stained with DAPI. Scale bar, 50 μm. Images are representative of a minimum of three independent biological replicates.
(C) qRT-PCR analysis of CD77, CD200, and SIRPA gene expression in human 15-week fetal atrial and ventricular tissue. Data were normalized to corresponding GAPDH expression.
Stem Cell Reports  , DOI: ( /j.stemcr )
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